Hyperoxia Elevates Adrenic Acid Peroxidation in Marine ...
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Title Hyperoxia Elevates Adrenic Acid Peroxidation in Marine Fishand Is Associated with Reproductive Pheromone Mediators
Author(s) CHUNG, ML; Galano, JM; Oger, C; Durand, T; Lee, CYJ
Citation Marine Drugs, 2015, v. 13 n. 4, p. 2215-2232
Issued Date 2015
URL http://hdl.handle.net/10722/209371
Rights Creative Commons: Attribution 3.0 Hong Kong License
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Mar. Drugs 2015, 13, 2215-2232; doi:10.3390/md13042215
marine drugs ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Hyperoxia Elevates Adrenic Acid Peroxidation in Marine Fish
and Is Associated with Reproductive Pheromone Mediators
Ming Long Sirius Chung 1, Jean-Marie Galano 2, Camille Oger 2, Thierry Durand 2 and
Jetty Chung-Yung Lee 1,*
1 School of Biological Sciences, the University of Hong Kong, Pokfulam Road, Hong Kong;
E-Mails: [email protected] (M.L.S.C.); [email protected] (J.C.-Y.L.) 2 Institute of Biomolecules Max Mousseron (IBMM), UMR 5247 CNRS, ENSCM, University of
Montpellier, F-34093 Montpellier cedex 05, France; E-Mails: [email protected] (J.-M.G.);
[email protected] (C.O.); [email protected] (T.D.)
* Author to whom correspondence should be addressed; E-Mail: [email protected];
Tel.: +852-2299-0318; Fax: +852-2559-9114.
Academic Editor: Gilles Barnathan
Received: 5 February 2015 / Accepted: 1 April 2015 / Published: 14 April 2015
Abstract: The development of oxidative stress in the marine ecosystem is a concurring
concern in fish reproductive behavior. Marine fish being rich in polyunsaturated fatty acids
(PUFA) are precursors of prostaglandin pheromone mediators but also vulnerable to lipid
peroxidation. It is yet to be determined if hypoxia or hyperoxia environment, a cumulative
effect in the marine ecosystem affect pheromone mediators in fish, and to understand if
this is associated with the generation of oxidized lipid products of PUFA. Novel oxidized
lipid metabolites, isoprostanoids (15-F2t-isoprostane, 7(RS)-7-F2t-dihomo-isoprostane,
17(RS)-17-F2t-dihomo-isoprostane, 8-F3t-isoprostane, 4(RS)-4-F4t-neuroprostane,
10-F4t-neuroprostane), isofuranoids (isofurans, 10-epi-17(RS)-SC-Δ15-11-dihomo-isofuran
and neurofurans), hydroxyeicosatetraenoic acids and resolvins, PUFA (arachidonic, adrenic,
eicosapentaenoic and docosahexaenoic acids) and prostaglandin pheromone mediators in fish
muscle were determined in marine male and female fish muscles before and after interaction in a
hypoxia or hyperoxia environment. Reproductive behaviors were also assessed. Our study
showed oxidized lipid metabolites of arachidonic, eicosapentaenoic, and docosahexaenoic
acids were not influenced by hypoxia and hyperoxia exposure in the fishes and no
gender differences were found. However, adrenic acid and its oxidized products,
17(RS)-17-F2t-dihomo-isoprostane and 10-epi-17(RS)-SC-Δ15-11-dihomo-isofuran showed
OPEN ACCESS
Mar. Drugs 2015, 13 2216
strong correspondence with male fish pheromone mediators and reproductive behavior when
under oxidative stress especially, hyperoxia. The occurrence of hypoxia and hyperoxia in the
marine ecosystem may not be detrimental to marine fish and instead presents as being
beneficial in reproductive behavior.
Keywords: adrenic acid; dihomo-isofurans; dihomo-isoprostanes; medaka
1. Introduction
The occurrence of oxidative stress by means of hypoxia and hyperoxia is a common phenomenon in
the marine ecosystem and under adverse conditions such as climate change and environmental
contaminants. Hypoxia condition in the marine ecosystem leads to excessive growth of some toxic algae
and disrupts sex hormone concentrations [1]. An overgrowth of these algae leads to death and
accumulation of organic matter by bacterial decomposition, and consequently causes oxygen
depletion [1–3]. Hyperoxia environment is a natural occurrence of photosynthetic organisms and the
presence of algae in the aquatic environment upon sunlight exposure elevates the rates of oxygen
production and excessive reactive oxygen species (ROS) accumulation [4,5].
Despite marine fish being rich in polyunsaturated fatty acids (PUFA), in particular docosahexaenoic
acid (DHA) and eicosapentaenoic acid (EPA), it is unclear if hypoxia and hyperoxia conditions lead to lipid
peroxidation. Exposure to chemical oxidants of marine fish acutely depleted PUFA and prostaglandin-F2α
(PGF2α) from arachidonic acid (AA), and elevated non-enzymatic oxidized lipid products mainly related
to PGF2α namely F2-isoprostanes from AA, F3-isoprostanes from EPA, F4-isoprostanes (also related as
F4-neuroprostanes) from DHA, and lipoxygenase-dependent lipid products [6]. However, PGF2α and its
derivatives, 15-keto-PGF2α and 13,14-dihydro-15-keto-PGF2α are also mediators for pheromone
production in fish muscles [7–10]. These pheromones are chemicals secreted externally by one individual
and received by a second individual. They serve for communication within species and may affect the
behavior and developmental process. The release of these pheromones can stimulate the central nervous
system and hence elevate the reproductive behavior of the fish [11]. Insufficient study has been made to
evaluate the effect and the role of these PUFAs and the oxygenated metabolites in marine fish development. It
is not only the yield of important essential PUFA in fish that may be affected by environmental oxidative
stress conditions, but it could impose physiological change in fish behavior and alter reproduction [1].
We investigated if hypoxia and hyperoxia conditions modify lipid peroxidation of omega-3 PUFA
(DHA and EPA) and omega-6 PUFAs (AA and adrenic acid, AdA), as well as the effect of eicosanoid
pheromone production in marine fish muscle.
2. Results
2.1. Effect of Oxidative Stress on Eicosanoid Pheromone Mediators
Exposure to hypoxia or hyperoxia to the fishes did not affect the generation of the pheromone
mediator PGF2α and the derivatives 15-keto-PGF2α and 13,14-dihydro-15-keto-PGF2α (Figure 1) in fish
Mar. Drugs 2015, 13 2217
muscles. The levels of pheromones did not show any differences between male and female fishes under
all treatment conditions.
Figure 1. Concentrations of prostaglandin pheromone mediators determined in fish muscles
under oxidative stress. Levels are mean ± SEM, n = 8 for all and n = 4 for each gender group.
2.2. Effect of Oxidative Stress on Fatty Acids
The impact of oxidative stress on omega-3 PUFAs (DHA and EPA) and omega-6 PUFAs
(AA and AdA) was not distinct. Hypoxia exposure for 1 h lowered AA and EPA levels but not DHA and
AdA in fish muscle compared to normoxia, and no effect was found after six hours exposure for all the
studied PUFAs (Figure 2). The exposure of one hour hyperoxia had no effect on all PUFAs compared to
normoxia but after six hours, only EPA reduced (Figure 2) in our study. We found only in hypoxia
environment showed decreased AA in male fish but no difference was found for other PUFAs between
male and female fish under all treatment conditions.
Mar. Drugs 2015, 13 2218
Figure 2. Concentrations of polyunsaturated fatty acids determined in fish muscles under
oxidative stress. Annotations indicate AA: arachidonic acid; AdA: adrenic acid;
EPA: eicosapentaenoic acid; DHA: docosahexaenoic acid. Levels are mean ± SEM, n = 8 for
all and n = 4 for each gender group. Only significant p-values tested by unpaired Student’s
t-test are annotated in the graphs. ANOVA indicates * p < 0.05 and ** p < 0.01 between
treatment and time.
2.3. Effect of Oxidative Stress on Generation of Lipid Peroxidation Products
2.3.1. Enzyme-Independent
Both hypoxia and hyperoxia treatments did not significantly change levels of oxidized products of
AA namely 15-F2t-isoprostane, 2,3-dinor-15-F2t-isoprostane and 2,3-dinor-5,6-dihydro-15-F2t-isoprostane
in fish muscles after one hour and six hours exposure compared to normoxia (Table 1). Hypoxia
treatment also did not show any effect on the release of the oxidized products, particularly
7(RS)-7-F2t-dihomo-isoprostane and 17(RS)-17-F2t-dihomo-isoprostane from AdA, 8-F3t-isoprostane from
Mar. Drugs 2015, 13 2219
EPA, and 4(RS)-4-F4t-neuroprostane and 10-F4t-neuroprostane from DHA compared to normoxia in fish
muscles after one hour and six hours exposure. In hyperoxia environment, the release of these oxidized
products did not alter compared to normoxia after one hour and six hours exposure. Furthermore in
normoxia, female fish muscle had higher 10-F4t-neuroprostane than male and no difference was found
between the genders under other treatment conditions (Table 1).
2.3.2. Enzyme-Dependent
Oxidized lipid products from AA could be released via lipoxygenase enzyme (LOX) to give 5(S)-,
8(S)-, 12(S)- and 15(S)-hydroxyeicosatetraenoic acid (HETE), from EPA to give resolvin E1 (RvE1) and
DHA to give resolvin D1 (RvD1). Only hypoxia exposure showed significant increased 5(S)-HETE
levels in male fishes and no gender difference was found between male and female fishes on other HETE
levels under all treatment conditions. Aside from 15(S)-HETE being elevated significantly after six
hours hyperoxia exposure, both hypoxia or hyperoxia treatment did not alter all the HETEs compared to
normoxia in fish muscle (Table 2). Hyperoxia condition altered RvE1 levels in the fish muscle
and no effect was found for RvD1 levels. There was no gender difference in RvD1 and RvE1 levels in
both treatments.
As displayed in Figure 3 both hypoxia and hyperoxia treatments did not affect the generation of
isofurans and neurofurans in fish muscles compared to normoxia. Moreover, these levels showed no
difference between male and female fishes at normoxia, and even when exposed to hypoxia and
hyperoxia conditions. However, it was intriguing to find hyperoxia exposure substantially augmented
10-epi-17(RS)-SC-Δ15-11-dihomo-isofuran level after six hours exposure compared to normoxia
environment, but not for hypoxia. It was identified that the elevation was significantly contributed by
male fishes and not by female fishes (Figure 3).
2.4. Correlation between PUFA and Oxidized Lipid Products, and Pheromone Mediators
It appears that the role of AdA is vital in the generation of pheromone mediators under oxidative
stress. Under hypoxia condition, strong positive correlation was identified between AdA and the
pheromone mediators in male and female fishes (Figure 4). However in hyperoxia condition, the
enzyme-independent oxidized products of AdA namely 17(RS)-17-F2t-dihomo-isoprostane and
10-epi-17(RS)-SC-Δ15-11-dihomo-isofuran had a strong association with pheromone mediators in male
fishes whereas a negative association was recorded for female fishes (Figure 4). No significant
correlation was found between AA, EPA or DHA, and the pheromone mediators, and the generation of
enzyme-dependent oxidized lipid products and the pheromone mediators.
Mar. Drugs 2015, 13 2220
Table 1. Concentrations of isoprostanoids from non-enzymatic peroxidation of polyunsaturated fatty acids in fish muscles after hypoxia and
hyperoxia exposure.
Normoxia Hypoxia Hyperoxia
Time (h) 0 1 6 p-trend 1 6 p-trend
Arachidonic Acid
15-F2t-IsoP
All
M
F
2.20 ± 0.76
1.51 ± 0.58
2.88 ± 1.43
2.44 ± 1.49
3.46 ± 2.90
3.41 ± 1.40
2.93 ± 0.90
2.33 ± 0.65
3.54 ± 1.77
0.727
0.738
0.951
1.26 ± 0.26
1.24 ± 0.39
1.29 ± 0.40
5.68 ± 2.24
7.24 ± 4.23
4.12 ± 1.94
0.078
0.210
0.398
2,3-dinor-15-F2t-IsoP+
All
M
F
1.89 ± 1.03
2.68 ± 2.13
1.10 ± 0.14
0.32 ± 0.06
0.27 ± 0.02
0.36 ± 0.12
0.76 ± 0.27
0.80 ± 0.32
0.72 ± 0.47
0.202
0.394
0.258
0.94 ± 0.25
1.24 ± 0.38
0.64 ± 0.29
0.63 ± 0.20
0.57 ± 0.20
0.72 ± 0.47
0.349
0.505
0.504
2,3-dinor-5,6-dihydro-15-F2t-IsoP+
All
M
F
19.43 ± 8.34
25.01 ± 16.30
13.16 ± 6.34
7.54 ± 1.39
7.95 ± 2.18
7.13 ± 2.03
7.96 ± 0.75
7.98 ± 1.44
7.95 ± 0.73
0.177
0.384
0.440
23.00 ± 8.27
29.00 ± 14.67
17.00 ± 8.94
13.84 ± 4.31
16.85 ± 8.58
10.83 ± 2.61
0.670
0.816
0.803
Adrenic Acid
7(RS)-7-F2t-dihomo-IsoP
All
M
F
0.98 ± 0.30
0.81 ± 0.43
1.17 ± 0.46
0.47 ± 0.09
0.49 ± 0.19
0.45 ± 0.06
0.50 ± 0.09
0.36 ± 0.09
0.64 ± 0.13
0.118
0.537
0.218
0.63 ± 0.15
0.68 ± 0.22
0.59 ± 0.25
0.56 ± 0.13
0.79 ± 0.17
0.33 ± 0.11
0.321
0.952
0.198
17(RS)-17-F2t-dihomo-IsoP
All
M
F
3.44 ± 1.43
4.33 ± 2.97
2.55 ± 0.42
2.37 ± 0.37
2.43 ± 0.61
2.31 ± 0.52
3.05 ± 0.54
2.13 ± 0.34
3.98 ± 0.83
0.706
0.646
0.173
3.99 ± 0.87
4.95 ± 1.64
3.02 ± 0.44
5.78 ± 1.45
8.15 ± 2.31
3.44 ± 0.86
0.409
0.501
0.604
Eicosapentaenoic Acid
8-F3t-IsoP
All
M
F
2.95 ± 0.53
3.03 ± 0.90
2.86 ± 0.70
2.58 ± 0.57
2.51 ± 1.05
2.65 ± 0.63
2.37 ± 0.33
2.65 ± 0.63
2.10 ± 0.23
0.705
0.912
0.626
2.75 ± 0.64
3.77 ± 0.98
1.74 ± 0.52
3.73 ± 0.92
4.48 ± 1.58
2.99 ± 1.04
0.598
0.701
0.492
Docosahexaenoic Acid
4(RS)-4-F4t-NeuroP
All
M
F
61.07 ± 11.80
54.21 ± 7.74
70.95 ± 6.41
71.44 ± 28.17
73.51 ± 49.02
69.37 ± 36.00
33.77 ± 6.79
37.96 ± 12.63
29.58 ± 6.63
0.284
0.706
0.347
61.07 ± 11.80
59.27 ± 19.80
62.87 ± 15.98
77.33 ± 26.96
97.96 ± 50.16
56.72 ± 24.35
0.766
0.580
0.845
10-F4t-NeuroP
All
M
F
1.54 ± 0.21
1.06 ± 0.13
2.01 ± 0.22 **
1.64 ± 0.50
0.94 ± 0.32
2.34 ± 0.85
0.94 ± 0.32
1.21 ± 0.59
0.68 ± 0.27
0.356
0.894
0.119
1.59 ± 0.25
1.35 ± 0.35
1.85 ± 0.37
1.01 ± 0.22
1.04 ± 0.44
0.99 ± 0.16
0.164
0.769
0.049
Values are mean ± SEM ng/g muscle except + μg/g muscle, n = 8 for all and n = 4 for each gender group. M: male; F: female; IsoP: isoprostane; NeuroP: neuroprostane; One-way ANOVA for linear p-trend is annotated
for the effect of oxygen tension change with time. ** p < 0.01 M vs. F.
Mar. Drugs 2015, 13 2221
Table 2. Concentration of lipoxygenase-mediated oxidized lipid products of polyunsaturated fatty acids in fish muscles after hypoxia and
hyperoxia exposure.
Normoxia Hypoxia Hyperoxia
Time (h) 0 1 6 p-trend 1 6 p-trend
Arachidonic Acid
5(S)-HETE
All
M
F
41.90 ± 4.98
29.62 ± 2.17
54.17 ± 12.12
31.05 ± 11.44
16.19 ± 3.63
45.91 ± 21.21
39.10 ± 7.06
40.53 ± 8.07
37.66 ± 12.87
0.672
0.029
0.771
26.20 ± 4.98
30.10 ± 9.16
22.29 ± 4.66
49.40 ± 12.38
52.25 ± 16.99
46.54 ± 20.53
0.188
0.312
0.294
8(S)-HETE
All
M
F
25.70 ± 5.66
26.41 ± 7.29
25.00 ± 9.78
26.08 ± 7.66
17.72 ± 7.72
34.43 ± 12.94
11.19 ± 1.93
10.84 ± 2.99
11.54 ± 2.90
0.126
0.274
0.282
30.79 ± 4.43
33.95 ± 7.62
27.64 ± 5.18
21.88 ± 3.96
25.92 ± 7.59
17.85 ± 2.14
0.425
0.707
0.566
12(S)-HETE
All
M
F
161.96 ± 62.88
173.00 ± 90.36
150.9 ± 101.00
157.03 ± 50.60
127.50 ± 47.59
186.50 ± 95.41
69.20 ± 19.23
73.66 ± 26.64
64.73 ± 31.65
0.325
0.538
0.579
203.29 ± 48.19
246.30 ± 91.76
160.30 ± 34.63
180.55 ± 53.86
259.30 ± 94.76
101.80 ± 20.51
0.870
0.756
0.783
15(S)-HETE
All
M
F
2.53 ± 0.29
2.66 ± 0.40
2.40 ± 0.48
13.00 ± 6.10
127.50 ± 47.59
186.50 ± 95.41
5.44 ± 2.55
7.63 ± 5.09
3.25 ± 1.08
0.160
0.543
0.332
1.79 ± 0.32
1.75 ± 0.45
1.82 ± 0.52
12.23 ± 4.85 *
15.33 ± 8.74
9.13 ± 5.18
0.027
0.781
0.209
Eicosapentaenoic Acid
RvE1
All
M
F
0.32 ± 0.04
0.26 ± 0.04
0.39 ± 0.07
0.21 ± 0.07
0.12 ± 0.01
0.30 ± 0.12
0.33 ± 0.12
0.28 ± 0.16
0.38 ± 0.20
0.533
0.446
0.894
0.17 ± 0.03
0.13 ± 0.05
0.20 ± 0.04
0.23 ± 0.04
0.27 ± 0.05
0.19 ± 0.05
0.027
0.142
0.045
Docosahexaenoic Acid
RvD1
All
M
F
1.15 ± 0.15
1.02 ± 0.22
1.28 ± 0.20
0.71 ± 0.11
0.66 ± 0.12
0.76 ± 0.20
2.91 ± 1.69
1.88 ± 0.90
3.95 ± 3.43
0.265
0.304
0.505
0.83 ± 0.09
0.82 ± 0.11
0.85 ± 0.15
0.72 ± 0.15
0.69 ± 0.26
0.75 ± 0.19
0.073
0.550
0.132
Values are mean ± SEM ng/g muscle n = 8 for all and n = 4 for each gender group. M: male, F: female. One-way ANOVA for linear p-trend is annotated for the effect of oxygen tension change
with time. HETE: hydroxyeicosatetraenoic acid; RvEI: resolvin E1; RvD1: resolvin D1. * p < 0.05 1 h vs. 6 h and normoxia vs. 6 h.
Mar. Drugs 2015, 13 2222
Figure 3. Concentrations of isofuranoids determined in marine fish muscles under oxidative
stress. Sources of isofurans are from AA, 10-epi-17(RS)-SC-Δ15-11-dihomo-isofuran from
AdA and neurofurans from DHA. Levels are mean ± SEM, n = 8 for all and n = 4 for each
gender group. Only significant p-values tested by unpaired Student’s t-test are annotated in
the graphs. ANOVA indicates ** p < 0.01 between treatment and time.
Mar. Drugs 2015, 13 2223
Hypoxia Hyperoxia
Figure 4. Pearson’s correlation of adrenic acid (AdA) and enzyme-independent oxygenated
metabolites, and pheromone mediators after hypoxia and hyperoxia exposure. Each point
represents one fish.
Mar. Drugs 2015, 13 2224
2.5. Effect of Hypoxia-Hyperoxia on Reproductive Behavior and Discharge of Pheromone
Mediators in Water
In order to test the reproductive behaviors under oxidative stress two actions, the pecking gestures
and following movements of the marine fishes were investigated. Both hypoxia and hyperoxia
conditions increased reproductive behavior (Figure 5A) compared to normoxia. However, in hyperoxia,
the male fishes contributed to 85% of the movements. Visual observations also showed sluggish
movement of the fishes after hypoxia treatment, whereas aggressive movements especially of male fish
were shown after hyperoxia treatment compared to the normoxic state. This behavior did not appear to
be related to the pheromone mediators released in the water. As shown in Figure 5B, change in oxygen
tension did not alter the concentration of free pheromone mediators released in the water after six hours
of treatment.
Figure 5. Reproductive behaviors (A) and pheromone mediator concentrations (B) in
marine water after exposure to oxidative stress. Male (M) and female (F) fishes were
contained in the same tank and exposed to hypoxia or hyperoxia for 6 h. Values (n = 4 pairs)
indicate number of incidences within one hour after exposure. F→M: female approach male;
M→F: male approach female; N: normoxia; H−: hypoxia; H+: hyperoxia. 2-test indicates
significant ** p < 0.01 vs. normoxia and †† p <0.01 M→F vs. normoxia and hypoxia.
A
B
All M→F F→M0
20
40
60
Nu
mb
er o
f In
cid
ence
s w
ith
in
1 h
ou
r aft
er e
xp
osu
re
Reproductive Behavior
N
H-
H+**
**
**
††
PGF2α 15-keto-PGF2α 13,14-dihydro-15-keto-PGF2α
0.00
0.02
0.04
0.06
0.1
0.2
0.3
0.4
Con
cen
trati
on
(n
g/m
l)
Pheromone Mediators in Water
N
H+
H-
Mar. Drugs 2015, 13 2225
3. Discussion
In this study we evaluated the effect of hypoxia and hyperoxia environments on PUFA metabolism in
marine fish. This is a follow-up study of our previous findings in which the presence of exogenous
hydrogen peroxide in marine water reduced PGF2α and PUFA levels, and induced production of oxidized
lipid products [6]. In this investigation we addressed the impact of oxidative stress on important
omega-3 and omega-6 PUFAs in marine fish by measuring novel oxidized lipid products. These
products included isoprostanoids, the PGF2α pheromone mediators and its derivatives, and more recently
identified isofuranoids, which have come to attention for their dominance in the hyperoxia environment
in vivo [12,13].
Evaluation of oxidized lipid products particularly isoprostanoids as biomarkers for in vivo assessment
is well defined and most of them are generated by non-enzymatic related ROS reaction with PUFA [13,14].
This biomarker of oxidative stress has been well studied in humans where AA is the common omega-6
PUFA in the body and DHA is probably the most omega-3 PUFA concentrated in the brain. However in fish
body, this might be different, since one of the most common PUFAs is DHA and with low concentration
of AA and probably AdA. Nevertheless in humans, it is common to assess products of AA i.e.,
15-F2t-isoprostane [7,14] whereas for more specialized conditions such as neurological disorders,
4(RS)-4-F4t-neuroprostane from DHA and 17(RS)-17-F2t-dihomo-isoprostane from AdA is
assessed [12,13,15,16]. Furthermore, it is known under high oxygen tension, release of isofurans from
AA [17,18], 10-epi-17(RS)-SC-Δ15-11-dihomo-isofuran from AdA [19] and neurofurans from
DHA [20] predominate. Nonetheless to date, oxidized products of AdA have been insufficiently
investigated in different biological systems.
The dynamics of hypoxia and hyperoxia environment also depends on the cleanliness of the marine
water and the climate; environmental pollutants such as contaminants from fertilizers and industrial
waste, and global warming concomitantly disrupt the oxygen balance in marine water [1,4,21]. Our
study showed exogenous change of oxygen concentration in marine water was not as detrimental as
extrinsic chemical insult. It was surprising to find hypoxia or hyperoxia conditions did not influence the
generation of oxidized lipid products from PUFA mainly 15-F2t-isoprostane, 8-F3t-isoprostane,
4(RS)-4-F4t-neuroprostane, and a majority of LOX-mediated products in the fish. The findings were
opposed to our previous report where exposure to a chemical oxidant such as hydrogen peroxide induced
oxidized lipid products and depleted its precursor PUFA [6]. Furthermore, only recently F2-isoprostanes
and neuroprostanes levels were assessed in the brain of zebrafish exposed to acute hypoxia but no
significant change was reported [22].
It should be noted that the generation of PGF2α and the derivatives in fish are associated with
pheromones produced in the ovaries or reproductive organs and released into the water. However, the
role of the prostaglandins is not limited to these, and in part act as a mediator for hormonal regulation in
reproduction and smooth muscle contraction. In our study, it was anticipated that the release of
prostaglandin pheromone mediators would be modified by the hypoxia and hyperoxia environments
since it is known that the hypoxic state reduces the rate of fertility and hatching in medaka [1].
Instead such observation was not made, and rather the generation of AdA and its oxygenated
metabolites showed an impact on prostaglandin pheromone mediators. It is clear hyperoxia in
marine fish affected AdA metabolism. Specifically, the association of its oxidized lipid products
Mar. Drugs 2015, 13 2226
17(RS)-17-F2t-dihomo-isoprostane and 10-epi-17(RS)-SC-Δ15-11-dihomo-isofuran, and the pheromone
mediators in male fish stimulated active reproductive behaviors. This also concurs with the use of
hyperoxic environment in fish hatchery for enhancing survival and growth of larvae in the early stages of
development [23]. However in this study, only up to six hours treatment of hypoxia or hyperoxia
was given to the fish, therefore the outcome is not known after prolonged treatment or after a
constant change of water as how it would be in the actual marine ecosystem. Without doubt
17(RS)-17-F2t-dihomo-isoprostane, 10-epi-17(RS)-SC-Δ15-11-dihomo-isofuran and AdA appear to take
part in fish (patho)physiology in this study as well as in pig brain, and rat heart and brain as shown in our
previous studies [19,24]. Despite the relatively low concentration of AdA compared to AA, DHA and
EPA, it seems to be sensitive to enzyme-independent peroxidation and the metabolites generated
(Figure 6) greatly influenced the fish endocrine system.
Figure 6. Structure of adrenic acid and its enzyme-independent oxidation products,
7(RS)-7-F2t-dihomo-IsoP, 17(RS)-17-F2t-dihomo-IsoP and 10-epi-17(RS)-SC-Δ15-11-dihomo-IsoF
identified in fish muscle after oxidative stress exposure. IsoP: isoprostane; IsoF: isofuran.
It would be interesting to investigate if 17(RS)-17-F2t-dihomo-isoprostane or 10-epi-17(RS)-S
C-Δ15-11-dihomo-isofuran indeed contribute to the reproductive behavior by microinjecting these
compounds to the reproductive system of the male fish to test the pheromone effect towards female fish.
Moreover, it should also be noted that the role of Δ5-desaturase enzyme in PUFA metabolism [25] might
be inhibited in the omega-3 PUFA pathway as there is a tendency for decreased EPA under oxidative
stress or the rate of β-oxidation under hyperoxia is enhanced and used for energy during the swimming
activity. As a follow-up, it would be appropriate to understand the benefits of this phenomenon in the
epigenetic approach; induce repeated hyperoxia-normoxia-hyperoxia conditions to manipulate
pheromone production to observe the fish population growth and sustainability.
Mar. Drugs 2015, 13 2227
4. Materials and Methods
4.1. Treatment of Medaka Fish
Adult marine medaka fishes (Oryzias melastigma) 10 months old were maintained in fully aerated
water set at photoperiod of 14 h light/10 h dark, water temperature at 26 ± 1 °C, ammonia level
(0.010 ± 0.005 mg/L) and pH 8. The fishes were fed twice daily with frozen artemia and hormone-free
flake food (AX5, Aquatic Eco-Systems Inc., Apopka, FL, USA) prior to exposure to hypoxia or
hyperoxia, and the water was cleaned (50% changed) every 72 h.
Medaka fish (n = 8) were paired (1:1, male to female) per treatment and placed in separate water tanks
of 3 L marine water. A hypoxia condition (dissolved oxygen 2.0–3.0 mg/L) was established by pumping
nitrogen gas for 5 min and a hyperoxia condition (dissolved oxygen 8.0–9.0 mg/L) was established by
pumping air into water. The water oxygen level was assessed using the dissolved oxygen monitor
(Pinpoint II, American Marine Inc., Ridgefield, CT, USA). The medaka fishes were exposed to hypoxia
or hyperoxia conditions for 1 h and 6 h following our previous report [6]. A normoxia condition was
established by placing medaka fish in fish tanks without an air pump (dissolved oxygen 6.0–7.0 mg/L)
and served as control. After treatment in different oxygen levels, each medaka fish was anesthetized on
ice and then sacrificed. All the internal organs were removed. The body muscle was washed by
phosphate buffered saline (PBS, pH 7.4) with indomethacin and butylated hydroxytoluene (BHT), and
stored at −80 °C for further lipid analysis. Volumes (50 mL) of water samples were taken at 6 h time
points and stored at −80 °C for free prostaglandin pheromones analysis.
4.2. Sample Preparation for Lipid Extraction
In this study we measured the lipids of the body muscle and not the liver (the deposit for fats and
lipids), as we were also investigating the body pheromone mediators level after the treatments. The main
body muscle (0.6–0.8 g) excluding the head and internal organs was homogenized in Folch solution with
BHT (0.005%). Sodium chloride solution (2 mL, 0.9% w/v) was added to the homogenized samples
thereafter centrifuged at 800× g for 10 min at 4 °C. The lower organic layer was transferred to a glass
vial and the solvent was evaporated under a stream of nitrogen gas. The dried Folch extract was
hydrolyzed with PBS (pH 7.4) and potassium hydroxide (1 M) prepared in methanol (1:1) at room
temperature overnight in the dark. After hydrolysis 40 mM formic acid buffer, methanol, and
hydrochloric acid were added before solid phase extraction. For solid phase extraction (SPE), 60 mg
MAX columns (Oasis, Waters, Milford, MA, USA) was used to purify the hydrolyzed samples
according to the Chung et al. [6] method. In brief, the column was conditioned with 20 mM formic acid,
washed with 2% ammonium hydroxide, hexane and hexane with ethyl acetate (70:30) and thereafter
eluted with hexane, ethanol, and acetic acid mix (70.00:29.98:0.02). The solvent from the extracted
samples was evaporated under a stream of nitrogen gas and the dried extract was re-dissolved in
methanol containing internal standards obtained from Cayman Chemicals (Ann Arbor, MI, USA) and
IBMM (Montpellier, France) for fatty acids, oxidized lipid products, and pheromones analysis. The
same SPE regime was used for purifying 20 mL of marine water collected at the 6 h time point for
measurement of free prostaglandin pheromones.
Mar. Drugs 2015, 13 2228
4.3. Analysis of Oxidized Lipid Products of Fatty Acids
Oxidized lipid products, 15-F2t-isoprostane, 2,3-dinor-15-F2t-isoprostane,
2,3-dinor-5,6-dihydro-15-F2t-isoprostane, 8-F3t-isoprostane, 4(RS)-4-F4t-neuroprostane,
10-F4t-neuroprostane, 7(RS)-7-F2t-dihomo-isoprostane, 17(RS)-17-F2t-dihomo-isoprostane, isofurans,
10-epi-17(RS)-SC-Δ15-11-dihomo-isofuran, neurofurans, LOX-mediated HETEs and resolvins (RvD1
and RvE1), and PUFAs (AA, AdA, EPA and DHA) analyses were performed using a 1290 Infinity LC
system (Agilent, Santa Clara, CA, USA) according to [6,19] with modification. A Hillic C18 column
(2.4 mm particle size, 50 × 2.4 mm, Phenomenex, Torrance, CA, USA) was used and maintained at
30 °C. The mobile phase consisted of 0.1% acetic acid in water (A) and 0.1% acetic acid in methanol (B)
and was set to 100 μL/min flow rate. The mobile phase ran in a linear gradient from phase A 50% to
phase B 50% over 3 min, B 95% to A 5% for 0.5 min and B 55% to A 45% for 3 min. The sample
injection volume was 20 μL. A QTrap 3200 triple quadrupole mass spectrometer (MS/MS, Sciex
Applied Biosystems, Carlsbad, CA, USA) was operated in the negative atmospheric pressure chemical
ionization (APCI) mode with a source temperature of 500 °C and capillary temperature of 250 °C. The
declustering and entrance potential, and the collision energy were set according to our previous reports
to maximize the ion currents of the precursor to product ion dissociation. The spray voltage was set to
−4000 V and nitrogen gas was used as curtain gas.
The analytes were detected by MS/MS using multiple reactions monitoring (MRM). Quantitation of
each analyte was achieved by relating the peak area with its corresponding internal standard peak. Heavy
labeled isotope AA-d8, EPA-d5, DHA-d5, 15-F2t-isoprostane-d4, 4(RS)-4-F4t-neuroprostane-d4,
10-F4t-neuroprostane-d4, 5(S)-HETE-d8, 12(S)-HETE-d8, and 15(S)-HETE-d8 were used for quantitation
of the respective compounds. Concentration of 7(RS)-7-F2t-dihomo-isoprostane,
17(RS)-17-F2t-dihomo-isoprostane, 8-F3t-isoprostane, isofurans, 10-epi-17(RS)-SC-Δ15-11-dihomo-isofuran
and RvE1 were determined using 15-F2t-isoprostane-d4, neurofurans and RvD1 using
4(RS)-4-F4t-neuroprostane-d4, and 8(S)-HETE using 5(S)-HETE-d8 as internal standard and where
possible, the ratio of response to the heavy labeled isotope was pre-determined with the non-deuterated
pure compounds for final concentration calculation.
4.4. Pheromone Mediator Analysis
The muscle lipid and water extracts from solid phase extraction were tested for the pheromones,
PGF2α, 15-keto-PGF2α and 13,14-dihydro-15-keto-PGF2α concentrations using LC-MS/MS. Similar
settings of the LC-MS/MS were made as for oxidized lipid products except for the declustering and
entrance potentials, collision energy, and precursor and product ions of dissociation. Heavy labeled
isotope PGF2α-d4 and 13,14-dihydro-15-keto-PGF2α-d4 were used for quantitation of the respective
compounds, and PGF2α-d4 for 15-keto-PGF2α.
4.5. Reproductive Behavior
After treatment of hypoxia and hyperoxia exposure for 6 h, the behaviors of the marine fishes in male
and female pairs (n = 4 pairs) per treatment were video recorded (Sony, Tokyo, Japan) for 1 h and the
total number of behavioral occurrences was recorded. From the list of behaviors [12], two common types
Mar. Drugs 2015, 13 2229
were identified which included, following and pecking. For following behavior, a pair of fish orientates
toward each other and stays in parallel. Both swim slowly, approach, and tail each other. For pecking
behavior the pair of fish peck-up each other and toss up their belly by staying at the bottom with their
mouths to orientate under one another.
4.6. Statistical Analysis
Statistical analysis was performed by GraphPad Prism version 6.0 for Macintosh (GraphPad
Software, La Jolla, CA, USA). All values are expressed as mean ± SEM. Any significant changes on
analysis of variance (mixed model) for the treatment were further analyzed by unpaired Student’s t-test.
Pearson’s correlation was tested between the pheromones and the analytes in male and female marine
fish separately. Chi-square (2) test was adapted for the reproductive behavioral test.
5. Conclusions
This study displayed the generation of a comprehensive set of oxidized lipid products of omega-3
and omega-6 polyunsaturated fatty acids (PUFA) rich marine fish under natural environmental
oxidative stress exposure and its effect on pheromone mediators. It is conspicuous that AdA and its
metabolites 17(RS)-17-F2t-dihomo-isoprostane and 10-epi-17(RS)-SC-Δ15-11-dihomo-isofuran had an
impact on male fish pheromone mediation and reproductive behavior when under oxidative stress. Our
findings also further indicate the importance of AdA and the oxidized lipid products of PUFA in
biological systems in evaluating oxidative stress whether as a biomarker or as bioactive compounds.
Acknowledgments
We thank The University of Hong Kong Seed Funding Programme for Basic Research
(No. 104001590 and 104002391) for the generous support of this work.
Author Contributions
M.L.S.C. and J.C.-Y.L. conceived and designed the experiments; M.L.S.C. performed the
experiments; J.M.G. and J.C.Y.L. analyzed the data; C.O., J.-M.G. and T.D. synthesized standards for
LC-MS/MS; J.C.Y.L. wrote the paper.
Abbreviation
2,3-dinor-15-F2t-IsoP: 2,3-dinor-15-F2t-isoprostane
2,3-dinor-5,6-dihydro-15-F2t-IsoP: 2,3-dinor-5,6-dihydro-15-F2t-isoprostane
4(RS)-4-F4t-NeuroP: 4(RS)-4-F4t-neuroprostane
7(RS)-7-F2t-dihomo-IsoP: 7(RS)-7-F2t-dihomo-isoprostane
8-F3t-IsoP: 8-F3t-isoprostane
10-F4t-NeuroP: 10-F4t-neuroprostane
13,14-dihydro-15-keto-PGF2α: 13,14-dihydro-15-keto prostaglandin F2α
15-F2t-IsoP: 15-F2t-isoprostane
15-keto-PGF2α: 15-keto-prostaglandin F2α
Mar. Drugs 2015, 13 2230
17(RS)-17-F2t-dihomo-IsoP: 17(RS)-17-F2t-dihomo-isoprostane
10-epi-17(RS)-SC-Δ15-11-dihomo-IsoF: 10-epi-17(RS)-SC-Δ15-11-dihomo-isofuran
AA: arachidonic acid
AdA: adrenic acid
DHA: docosahexaenoic acid
EPA: eicosapentaenoic acid
HETE: hydroxyeicosatetraenoic acid products
IsoFs: isofurans
LC-MS/MS: liquid chromatography tandem mass spectrometry
LOX: lipoxygenase
NeuroFs: neurofurans
PGF2α: prostaglandin F2α
RvD1: resolvin D1
RvE1: resolvin E1
Conflicts of Interest
The authors declare no conflict of interest.
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